This proposal aims to determine the relationship between changes in synaptic structure and strength during synapse elimination at developing mammalian neuromuscular junctions (NMJ). During this period, competition between inputs leads to disparities in their size and strength and ultimately the withdrawal of all but one axon from the NMJ. To evaluate the hypothesis that the axon occupying the larger synaptic territory is also the stronger input that will emerge the winner in this competitive process, both optical and physiological measurements of synaptic function will be made at multiply innervated neonatal NMJs from mice that express variants of GFP in their motor axons. To determine the relationship between relative neurotransmitter release probability and the terminal size of competing inputs, fluorescence changes in competing motor axon terminals labeled with FM1-43 and FM4-64, dyes that are taken up and released by synaptic vesicles in an activity dependent manner, will be measured as the axons are stimulated. Comparisons of the size and overall synaptic strength of competing inputs will be made via intracellular recordings from single muscle fibers innervated by two axons expressing different fluorescent tags. Finally, to characterize the spatial and temporal changes in input size and strength, an in vivo imaging approach will be used to repeatedly visualize changes in terminal size and neurotransmitter release rates of FM1-43 labeled inputs at the same junction as synapse elimination occurs. These experiments will provide new information about the mechanisms underlying competition and how these culminate in synapse elimination.